Beta-carboline 3, 6, 9-position modified derivative as well as preparation method and application thereof

Abstract

The invention relates to a beta-carboline 3, 6, 9-position modified derivative as well as a preparation method and application thereof, and belongs to the technical field of natural medicines and medicinal chemistry. In particular to a beta-carboline 3, 6, 9-position modified derivative or pharmaceutically acceptable salt thereof, further provides a pharmaceutical composition, provides a preparation method of the beta-carboline 3, 6, 9-position modified derivative, and also provides application of the beta-carboline 3, 6, 9-position modified derivative in preparation of SIRT6 inhibitors or tumor treatment drugs. The beta-carboline 3, 6, 9-position modified derivative or the pharmaceutically acceptable salt thereof disclosed by the invention is shown as a general formula I in the specification. Wherein X, n, R1, R2 and R3 are as described in the claims and the specification. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a β-carboline 3,6,9-position modified derivative and a preparation method and application thereof, belonging to the technical field of natural medicine and medicinal chemistry. Background Art

[0002] SIRT6 belongs to the family of class III histone deacetylases, mediating the deacetylation of histones and non-histones. In addition, SIRT6 can promote the deacylation of long-chain fatty acid groups and has ADP ribosyltransferase activity. Abnormally high expression of SIRT6 is associated with poor prognosis in a variety of tumors. SIRT6 promotes the progression of breast cancer by enhancing oxidative phosphorylation and induces tumor resistance by enhancing DNA damage repair mechanisms. Although some compounds show inhibitory potential for SIRT6, the exploration of SIRT6 inhibitors is still in its early stages. Pharmacological inhibition of SIRT6 has shown its advantages in anti-tumor and type 2 diabetes treatment. Although some compounds show certain SIRT6 inhibitory potential, there are still problems such as lack of subtype selectivity and poor drugability. It is still necessary to find new potential SIRT6 inhibitors. Summary of the invention

[0003] The present invention provides a 3,6,9-modified derivative of β-carboline and a preparation method and use thereof. The purpose is to provide a 3,6,9-modified derivative of β-carboline or a pharmaceutically acceptable salt thereof, and further provide a pharmaceutical composition, provide a preparation method of the 3,6,9-modified derivative of β-carboline, and point out the use of the 3,6,9-modified derivative of β-carboline in the preparation of SIRT6 inhibitors or the preparation of drugs for treating tumors.

[0004] Specifically, the present invention provides the following technical solutions:

[0005] The first object of the present invention is to provide a β-carboline 3,6,9-modified derivative or a pharmaceutically acceptable salt thereof, wherein the general structural formula of the β-carboline 3,6,9-modified derivative is as follows:

[0006]

[0007] in,

[0008] X is O or NH, n is 1 or 2;

[0009] R 1 is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the heterocyclic ring in the heteroaryl contains 1-4 N, O or S atoms, and the substituent in the substituted heteroaryl is a halogen, or a hydroxyl, or a methoxy, or an alkyl group containing 1-7 carbon atoms;

[0010] R 2 is an aryl or substituted aryl group containing 4-13 carbon atoms, or a hydrocarbon group containing 1-10 carbon atoms, or a cycloalkyl group containing 3-5 carbon atoms, or an alkoxy group containing 1-4 carbon atoms, or a heteroaryl or substituted heteroaryl group containing 2-10 carbon atoms, wherein the aromatic heterocyclic ring in the heteroaryl group contains 1-4 N, O or S atoms as heteroatoms, and the substituent in the substituted aryl or substituted heteroaryl group is halogen, or hydroxyl, or cyano, or trifluoromethyl, or methoxy, or trifluoromethoxy, or an alkyl group containing 1-8 carbon atoms;

[0011] R 3 It is hydrogen, or halogen, or methoxy, or an alkyl group containing 1 to 9 carbon atoms.

[0012] The abovementioned heteroaryl radicals may be derived from a heterocycle containing one, two, three or four identical or different ring heteroatoms, in particular from a heterocycle containing one, two or three, in particular one or two identical or different heteroatoms, which heterocycle may be mono- or polycyclic, such as mono-, bi- or tricyclic, preferably mono- or bicyclic, in particular monocyclic.

[0013] The aryl group mentioned above is derived from an aromatic ring, and may be a mono- or polycyclic ring, such as a mono-, bi- or tri-cyclic ring, preferably a mono- or bi-cyclic ring.

[0014] The substituted heteroaryl or substituted aryl mentioned above may be monosubstituted or polysubstituted. In the case of monosubstituted, the substituent may be in any position. In the case of polysubstituted, the substituent may be the same or different and may be in any position.

[0015] The halogen mentioned above refers to a fluorine, chlorine, bromine or iodine atom.

[0016] Preferably, X is NH, and n is 1 or 2.

[0017] More preferably, X is NH, and n is 1.

[0018] Preferably, the R 1 It is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the aromatic heterocyclic ring in the heteroaryl contains 1, 2 or 3 heteroatoms of N, O or S atoms, and the substituent in the substituted heteroaryl is halogen, or hydroxyl, or an alkyl group containing 1 to 4 carbon atoms.

[0019] More preferably, the R 1 It is pyridyl, furyl, thienyl, triazolyl, pyrazinyl, pyrimidinyl, thiazolyl, wherein the substituent is halogen, or hydroxyl, or methyl.

[0020] Most preferably, R 1 It is pyridyl.

[0021] Preferably, the R 2 It is phenyl or substituted phenyl, or naphthyl, or alkenyl containing 2-4 carbon atoms, or cycloalkyl containing 3 or 4 carbon atoms, or alkoxy containing 2 or 3 carbon atoms, wherein the substituent in the substituted phenyl is halogen, or cyano, or trifluoromethyl, or methoxy, or trifluoromethoxy, or alkyl containing 1-4 carbon atoms.

[0022] More preferably, the R 2 It is phenyl or substituted phenyl, or naphthyl, or isobutylene, or cyclopropane, or ethoxy, wherein the substituent in the substituted aryl is a fluorine atom, or a chlorine atom, or a cyano group, or a trifluoromethyl group, or a methoxy group, or a trifluoromethoxy group, or a tert-butyl group.

[0023] Most preferably, the R 2 is a substituted phenyl group, wherein the substituent is a cyano group.

[0024] Preferably, the R 3 It is a hydrogen, a chlorine, or a bromine atom.

[0025] Most preferably, the R 3 A chlorine atom.

[0026] Particularly preferably, X is NH, n is 1, R 1 is pyridyl, R 2 is a substituted phenyl group, wherein the substituent is a cyano group, and R 3 A chlorine atom.

[0027] Furthermore, the structural formula of the 3,6,9-modified derivatives of β-carboline and their pharmaceutically acceptable salts are as follows:

[0028]

[0029] The second object of the present invention is to provide a method for preparing the above-mentioned β-carboline 3, 6, 9-modified derivatives or pharmaceutically acceptable salts thereof.

[0030] The β-carboline 3,6,9-position modified derivatives of the present invention can be prepared by the following method, comprising the following steps:

[0031] (1) Compound 1 L-tryptophan was dissolved in sodium hydroxide solution, 37% formaldehyde solution was added and the mixture was reacted at 37°C for 2 days to obtain compound 2; compound 2 was dissolved in anhydrous methanol, thionyl chloride was added dropwise under ice bath condition, and the mixture was refluxed at 70°C for 6 hours to obtain compound 3; compound 3 was dissolved in N,N-dimethylformamide solution, potassium permanganate was added under ice bath condition, the mixture was reacted for 1 hour, and then the mixture was transferred to room temperature for reaction for 14 hours to obtain compound 4.

[0032] (2) Compound 4 was dissolved in methanol, 1N NaOH solution was added, and the mixture was heated at 78°C for 2 h to obtain compound 5; compound 5 was dissolved in N,N-dimethylformamide solution, carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding amino group were added. The compound was obtained by reacting at room temperature for 12 hours. Or add carbodiimide hydrochloride, 4-dimethylaminopyridine and the corresponding alcohol Compound Dissolve the above compound in tetrahydrofuran solution containing sodium hydride, stir for 1 hour, then add the corresponding bromohydrocarbon or benzyl bromide. React at room temperature for 12 hours to obtain the compound or

[0033] Alternatively, the β-carboline 3, 6, 9-position modified derivatives of the present invention can be prepared by the following method, comprising the following steps:

[0034] (1) Compound 1 L-tryptophan was dissolved in a sodium hydroxide solution, and a 37% formaldehyde solution was added and reacted at 37°C for 2 days to obtain compound 2; compound 2 was dissolved in anhydrous methanol, thionyl chloride was added dropwise under an ice bath condition, and refluxed at 70°C for 6 hours to obtain compound 3; compound 3 was dissolved in N,N-dimethylformamide solution, potassium permanganate was added under an ice bath condition, and the reaction was transferred to room temperature for 14 hours to obtain compound 4;

[0035] (2) Compound 4 was dissolved in dichloromethane, a halogenating agent was added, and the mixture was refluxed at 78°C for 12 h to obtain compound The above compound was dissolved in methanol, 1N NaOH solution was added, and the mixture was heated at 78°C for 2 h to obtain the compound The above compounds were dissolved in N,N-dimethylformamide solution, carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding amino The compound was obtained by reacting at room temperature for 12 hours. Or add carbodiimide hydrochloride, 4-dimethylaminopyridine and the corresponding alcohol Compound Dissolve the above compound in tetrahydrofuran solution containing sodium hydride, stir for 1 hour, then add the corresponding bromohydrocarbon or benzyl bromide. React at room temperature for 12 hours to obtain the compound

[0036] In the above technical solution, the compound 1, compound 2, compound 3, compound 4 and compound 5 have the following structures respectively:

[0037]

[0038] In the above technical solution, the reaction order in step (2) is not important, and any order can achieve the purpose of the present invention.

[0039] Preferably, the corresponding amines are 2-methylaminopyridine, 2-methylaminothiophene, 2-methylaminofuran, 3-methylaminopyridine, 2-(2-aminoethyl)pyridine, 2-aminomethyl-5-chloropyridine, 2-aminomethyl-5-fluoropyridine, 6-methyl-2-pyridinemethylamine, 2-aminomethylpyrazine, and 4-aminomethylpyrimidine.

[0040] Preferably, the corresponding alcohols are 2-chloro-5-hydroxymethylpyridine, 2-hydroxymethylpyrazine, 5-hydroxymethylthiazole, 3-hydroxymethylpyridine, and 1-hydroxymethyl-1,2,4-triazole.

[0041] Preferably, the corresponding benzyl bromide or bromohydrocarbon is 4-tert-butylbenzyl bromide, 1-bromo-trifluoro-p-xylene, 4-chlorobenzyl bromide, 3,4,5-trifluorobenzyl bromide, 4-chloro-3-fluorobenzyl bromide, p-cyanobenzyl bromide, 2-(bromomethyl)naphthalene, bromomethylcyclopropane, 1-bromo-3-methyl-2-butene, 2-bromoethyl methyl ether, 2,3,6-trifluorobenzyl bromide, 4-fluoro-3-trifluoromethylbenzyl bromide, 4-trifluoromethoxybenzyl bromide, 3,5-dimethoxybenzyl bromide.

[0042] Preferably, the halogenating agent is N-chlorosuccinimide or N-bromosuccinimide.

[0043] Particularly preferably, the corresponding amine is dimethylaminopyridine, the corresponding benzyl bromide is 4-cyanobenzyl bromide, and the halogenating agent is N-chlorosuccinimide.

[0044] The third object of the present invention is to provide a pharmaceutical composition, which contains a therapeutically effective amount of the β-carboline 3,6,9-modified derivatives represented by the general formula and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.

[0045] The fourth object of the present invention is to provide the use of the 3,6,9-modified derivatives of β-carboline and pharmaceutically acceptable salts thereof or the pharmaceutical composition in the preparation of SIRT6 inhibitors.

[0046] The fifth object of the present invention is to provide the use of the β-carboline 3,6,9-modified derivatives and pharmaceutically acceptable salts thereof or the pharmaceutical composition in the preparation of drugs for treating tumor diseases.

[0047] Furthermore, the tumor is breast cancer, pancreatic cancer, leukemia, neuroblastoma, liver cancer, or lung cancer.

[0048] Furthermore, the breast cancer includes breast cancer cells MCF-7, MDA-MB-231, and MDA-MB-453; the pancreatic cancer includes pancreatic cancer cells BxPC-3, Panc-1, MIA-PaCa-2, and Capan-1; the leukemia includes leukemia cells HL-60, K562, U937, and THP-1; the neuroblastoma includes neuroblastoma cells SH-SY5Y; the liver cancer includes liver cancer cells HepG2, Huh7, and Hep3B; and the lung cancer includes lung cancer cells A549, BE1, and LH7.

[0049] Beneficial effects of the present invention: The 3, 6, and 9-position modified derivatives of β-carboline provided by the present invention have a good inhibitory effect on protein lysine deacetylation of SIRT6, and show good anti-tumor activity against tumor cells such as MCF-7, HL-60, SH-SY5Y, and MIA-PaCa-2, and have the potential to be used as anticancer drugs. DETAILED DESCRIPTION

[0050] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0051] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0052] The β-carboline 3,6,9-modified derivatives described in the following examples were prepared according to the following synthetic route:

[0053]

[0054]

[0055] Example 1

[0056] The structure of compound 10d is shown below:

[0057]

[0058] (1) 10 g of compound 1L-tryptophan (49.02 mmol) was dissolved in 10 mL of 0.5 N NaOH solution, and then 6 mL of 37% formaldehyde solution (73.94 mmol) was added and reacted at 37° C. for two days. The reaction was basically completed as monitored by thin layer chromatography (TLC). The mixture was cooled, and then glacial acetic acid was added to adjust the pH until a precipitate was formed. The mixture was filtered and dried to obtain 29.28 g of the compound.

[0059] (2) 9.28 g of compound 2 (42.96 mmol) was dissolved in 120 ml of anhydrous methanol and 7.36 mL of SOCl was added dropwise under ice bath conditions. 2 (101.34 mmol), then reflux at 70°C for 6 h. After TLC monitoring, the reaction was complete, cooled, concentrated, and then added with 100 mL of saturated sodium bicarbonate solution, extracted with ethyl acetate three times, washed with saturated brine once, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 36 g of compound.

[0060] (3) 6 g of compound 3 (30.44 mmol) was dissolved in 100 mL of DMF, and 14 g of potassium permanganate (88.58 mmol) was added under ice bath conditions. After reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h. After TLC monitoring, the reaction was complete, and the filtrate was filtered, extracted with ethyl acetate three times, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=30:1) to obtain 44.0 g of compound.

[0061] (4) 4 g of compound 4 (17.69 mmol) was dissolved in dichloromethane, and 2.36 g of N-chlorosuccinimide (17.69 mmol) was added. The mixture was refluxed at 78° C. overnight and monitored by TLC. When the reaction was complete, the mixture was filtered and the filtrate was spin-dried. The mixture was extracted with ethyl acetate three times, washed with saturated brine once, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=40:1) to obtain 3.5 g of compound 4a.

[0062] (5) 1 g of compound 4a (3.84 mmol) was dissolved in dichloromethane, and 10 mL of 1 N NaOH solution was added. The mixture was heated to react for 2 h. The reaction was complete as monitored by TLC. The mixture was cooled, and then 2 N hydrochloric acid solution was added to adjust the pH to 4-5. A precipitate was formed, which was filtered and dried to obtain 890 mg of compound 8a.

[0063] (6) 320 mg of compound 8a (1.3 mmol) was dissolved in DMF, and 320 mg (1.65 mmol) of carbodiimide hydrochloride (EDCI) and 225 mg (1.65 mmol) of 1-hydroxybenzotriazole (HOBT) were added. The mixture was stirred at room temperature for 30 min, and then 140.4 mg (1.3 mmol) of 2-aminomethylpyridine was added and reacted for 12 h. After TLC monitoring, the reaction was complete. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=100:1) to obtain 319 mg of compound 9a.

[0064] (7) 100 mg of compound 9a (0.29 mmol) was dissolved in anhydrous tetrahydrofuran, 90 mg of sodium hydride (3.75 mmol) was added, and the mixture was stirred at room temperature for 40 min, followed by the addition of 56.87 mg (0.29 mmol) of 4-cyanobenzyl bromide, and the mixture was reacted at room temperature for 2 h. After TLC monitoring, the reaction was complete, and the mixture was filtered off with suction, and the filtrate was spin-dried, extracted with ethyl acetate three times, washed with saturated brine twice, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=100:1) to obtain 2.91 g of compound 10d as a white solid, with a yield of 59%. 1 H NMR (CDCl 3 ,600MHz)δ8.97(1H,s),8.88(1H,d,J=1.0Hz),8.66(1H,d,J=1.0Hz),8.59(1H,dd d,J=4.9,1.8,0.9Hz),8.15(1H,d,J=2.0Hz),7.67(1H,td,J=7.7,1.8Hz),7.59–7 .55(2H,m),7.54(1H,dd,J=8.7,2.0Hz),7.37(1H,d,J=7.8Hz),7.31(1H,d,J=8.7 Hz),7.20–7.18(2H,m),7.18(1H,d,J=1.8Hz),5.64(2H,s),4.85(2H,d,J=5.6Hz). 13 CNMR (CDCl 3 ,151MHz)δ165.07,157.21,149.31,141.20,140.95,139.75,137.91,136.79,132.94,130.41,129.49,128.53,1 27.00,126.92,122.89,122.32,122.11,121.99,118.17,114.61,112.27,110.75,46.85,44.83.HRMS(ESI)calcd for C 26 H 19 C1N 5 O,[M+H] + :m / z452.1273, found 452.1274.

[0065] Example 2

[0066] The structure of compound 7a is shown below:

[0067]

[0068] (1) 10 g of compound 1L-tryptophan (49.02 mmol) was dissolved in 10 mL of 0.5 N NaOH solution, and then 6 mL of 37% formaldehyde solution (73.94 mmol) was added and reacted at 37° C. for two days. The reaction was basically completed as monitored by thin layer chromatography (TLC). The mixture was cooled, and then glacial acetic acid was added to adjust the pH until a precipitate was formed. The mixture was filtered and dried to obtain 29.28 g of the compound.

[0069] (2) 9.28 g of compound 2 (42.96 mmol) was dissolved in 120 ml of anhydrous methanol and 7.36 mL of SOCl was added dropwise under ice bath conditions. 2 (101.34 mmol), then reflux at 70°C for 6 h. After TLC monitoring, the reaction was complete, cooled, concentrated, and then added with 100 mL of saturated sodium bicarbonate solution, extracted with ethyl acetate three times, washed with saturated brine once, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 36 g of compound.

[0070] (3) 6 g of compound 3 (30.44 mmol) was dissolved in 100 mL of DMF, and 14 g of potassium permanganate (88.58 mmol) was added under ice bath conditions. After reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h. After TLC monitoring, the reaction was complete, and the filtrate was filtered, extracted with ethyl acetate three times, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=30:1) to obtain 44.0 g of compound.

[0071] (4) 1 g of compound 4 (4.42 mmol) was dissolved in dichloromethane, and 10 mL of 1 N NaOH solution was added. The mixture was heated to react for 2 h. The reaction was complete as monitored by TLC. The mixture was cooled, and then 2 N hydrochloric acid solution was added to adjust the pH to 4-5. A precipitate was formed, which was filtered and dried to obtain 5920 mg of the compound.

[0072] (5) 500 mg of compound 5 (2.36 mmol) was dissolved in DMF, and 543 mg (2.83 mmol) of carbodiimide hydrochloride (EDCI) and 600 mg (2.83 mmol) of 1-hydroxybenzotriazole (HOBT) were added. The mixture was stirred at room temperature for 30 min, and then 255 mg (2.36 mmol) of 2-aminomethylpyridine was added. The mixture was reacted for 12 h. After TLC monitoring, the reaction was complete. The reaction solution was extracted 3 times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=100:1) to obtain 292 mg of compound 6a.

[0073] (6) 100 mg of compound 6a (0.33 mmol) was dissolved in anhydrous tetrahydrofuran, 150 mg of sodium hydride (6 mmol) was added, and the mixture was stirred at room temperature for 40 min, followed by 64.7 mg of 4-tert-butylbenzyl bromide (0.33 mmol) and reacted at room temperature for 2 h. After TLC monitoring, the reaction was complete, and the filtrate was filtered and dried, extracted with ethyl acetate 3 times, washed with saturated brine 2 times, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH=100:1) to obtain 68 mg of compound 7a as a white solid with a yield of 49%. 1 H NMR (CDCl 3 ,600MHz)δ9.01–8.95(2H,m),8.75(1H,d,J=1.0Hz),8.60(1H,ddd,J=4.9,1.8,0.9Hz ),8.25(1H,dt,J=8.0,1.0Hz),7.66(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8.3,7.1 ,1.2Hz),7.53–7.47(1H,m),7.41–7.32(2H,m),7.32–7.27(2H,m),7.19(1H,ddd,J=7 .5,4.8,1.1Hz),7.11–7.06(2H,m),5.58(2H,s),4.87(2H,d,J=5.7Hz),1.26(9H,s). 13 CNMR (CDCl 3 ,151MHz)δ165.67,157.58,151.08,149.28,141.92,140.33,137.92,136.80,133.02,130.58,129.27,128.93,126.25,125.95,122 .36,122.24,122.00,121.75,120.72,114.54,109.98,77.26,77.04,76.83,46.88,44.89,34.53,31.26.HRMS(ESI)calculatedfor C 29 H 29 N 4 O,[M+H] + :m / z 449.2336, found 449.2335.

[0074] Example 3

[0075] The structure of compound 7f is shown below:

[0076]

[0077] Using 4-trifluoromethylbenzyl bromide to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2, and repeating the experimental procedures of Example 1 with other conditions unchanged, compound 7f was obtained as a white solid with a yield of 61.3%. 1 H NMR (CDCl 3 ,600MHz)δ9.01–8.95(2H,m),8.75(1H,d,J=1.0Hz),8.60(1H,ddd,J=4.9,1.8,0.9Hz ),8.25(1H,dt,J=8.0,1.0Hz),7.66(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8.3,7.1 ,1.2Hz),7.53–7.47(1H,m),7.41–7.32(2H,m),7.32–7.27(2H,m),7.19(1H,ddd,J=7 .5,4.8,1.1Hz),7.11–7.06(2H,m),5.58(2H,s),4.87(2H,d,J=5.7Hz),1.26(9H,s). 13 C NMR (CDCl 3 ,151MHz)δ165.67,157.58,151.08,149.28,141.92,140.33,137.92,136.80,133.02,130.58,129.27,128.93,126.25,125.95,1 22.36,122.24,122.00,121.75,120.72,114.54,109.98,77.26,77.04,76.83,46.88,44.89,34.53,31.26.HRMS(ESI)calculated forC 29 H 29 N 4 O,[M+H] + :m / z 449.2336, found 449.2335.

[0078] Example 4

[0079] The structure of compound 7k is shown below:

[0080]

[0081] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced by 4-chlorobenzyl bromide. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 7k as a white solid with a yield of 45.2%. 1 H NMR (CDCl 3,600MHz)δ9.01–8.96(2H,m),8.70(1H,d,J=1.0Hz),8.60(1H,dt,J=5.0,1.4Hz),8.24(1H,d,J=7.8Hz),7.69–7.63(1H,m),7.63–7.57(1H, m),7.43(1H,d,J=8.3Hz),7.41–7.34(2H,m),7.28–7.22(2H,m),7.22–7.16(1H,m),7.08–7.04(2H,m),5.56(2H,s),4.87(2H,d,J=5.7Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.54,157.47,149.26,141.68,140.60,137.74,136.83,134.52,133.92,130.29,129.41,129.2 5,129.10,127.83,122.46,122.28,122.00,121.80,120.99,114.57,109.80,46.56,44.86.HRMS(ESI)calcd for C 25 H 19 C1N 4 O,[M+H] + :m / z427.132,found427.1326.

[0082] Example 5

[0083] The structure of compound 7p is shown below:

[0084]

[0085] 3,4,5-trifluorobenzyl bromide was used to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2. The experimental procedures of Example 1 were repeated with other conditions unchanged to obtain compound 7p as a white solid with a yield of 66.2%. 1 H NMR (CDCl 3,600MHz)δ8.99(1H,t,J=5.7Hz),8.97(1H,d,J=0.9Hz),8.67(1H,d,J=1.0Hz) ,8.62–8.58(1H,m),8.26–8.21(1H,m),7.66(1H,td,J=7.6,1.8Hz),7.62(1H, td,J=7.6,7.1,1.2Hz),7.39(3H,ddd,J=7.4,4.5,2.0Hz),7.20(1H,ddd,J=7. 6,4.9,1.1Hz),6.73(2H,dd,J=7.7,6.2Hz),5.53(2H,s),4.87(2H,d,J=5.6Hz) 13 C NMR (CDCl 3 ,151MHz)δ165.40,157.39,149.27,141.42,140.99,137.54,136.84,129.97,129.95,129.62,129.34,122.60,1 22.31,122.01,121.87,121.33,114.63,110.64,110.61,110.53,110.50,109.51,46.02,44.86.HRMS(ESI)calcd for C 25 H 18 F 3 N 4 O,[M+H] + :m / z 447.1427,found447.1428.

[0086] Example 6

[0087] The structure of compound 7A is shown below:

[0088]

[0089] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced by 4-nitrilebenzyl bromide. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 7A as a white solid with a yield of 71.2%. 1 H NMR (CDCl 3,600MHz)δ8.98(2H,d,J=9.1Hz),8.67(1H,s),8.59(1H,dd,J=5.0,1.7Hz),8.24(1H,d,J=7.8Hz),7.66(1H,td,J=7.7,1.8Hz),7.61 (1H,td,J=7.7,7.0,1.2Hz),7.58–7.54(2H,m),7.42–7.36(3H,m),7.19(3H,dd,J=10.8,6.6Hz),5.65(2H,s),4.86(2H,d,J=5.6Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.41,157.37,149.31,141.53,141.41,140.93,137.63,136.81,132.89,130.06,129.56,129.29,1 27.08,122.57,122.31,122.00,121.86,121.27,118.28,114.61,112.11,109.60,46.71,44.86.HRMS(ESI)calcd for C 26 H 20 N 5 O,[M+H] + :m / z418.1663 found418.1658.

[0090] Example 7

[0091] The structure of compound 7B is shown below:

[0092]

[0093] Using 2-(bromomethyl)naphthalene to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2, and other conditions remaining unchanged, the experimental procedures of Example 1 were repeated to obtain compound 7B as a white solid with a yield of 49.2%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,s),8.97(1H,t,J=5.8Hz),8.77(1H,s),8.58(1H,dd,J=5.1,1.7Hz),8.26( 1H,d,J=7.8Hz),7.80–7.73(2H,m),7.70–7.66(1H,m),7.64(1H,td,J=7.7,1.8Hz),7.60(1H,d ,J=8.2Hz),7.57(1H,dd,J=6.7,1.4Hz),7.51(1H,d,J=8.2Hz),7.46–7.42(2H,m),7.37(2H,t, J=7.9Hz),7.28–7.23(1H,m),7.17(1H,dd,J=7.4,4.9Hz),5.75(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.63,157.51,149.22,141.92,140.43,137.99,136.80,133.43,133.29,132.95,130.49,129.37,129.07,129.02,127.7 8,127.73,126.58,126.27,125.33,124.23,122.40,122.23,121.96,121.81,120.85,114.56,110.00,47.46,44.85.HRMS(ESI)calcd for C 29 H 22 N 4 ONa,[M+Na] + :m / z465.1686,found465.1692.

[0094] Example 8

[0095] The structure of compound 7C is shown below:

[0096]

[0097] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced by bromomethylcyclopropane. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 7C as a white solid with a yield of 32.6%. 1 H NMR (CDCl 3,600MHz)δ9.02(1H,t,J=5.8Hz),8.94(1H,s),8.79(1H,s),8.61(1H,dd,J=5.1,1.7Hz),8.2 0(1H,d,J=7.8Hz),7.65(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8.2,6.9,1.2Hz),7.50(1H, d,J=8.3Hz),7.40(1H,d,J=7.8Hz),7.33(1H,t,J=7.5Hz),7.21–7.16(1H,m),4.88(2H,d,J= 5.6Hz), 4.29 (2H, d, J = 6.6Hz), 1.39–1.28 (1H, m), 0.61–0.55 (2H, m), 0.42 (2H, q, J = 5.3Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.76,157.63,149.30,141.73,139.92,137.86,136.77,130.36,128.96,128.72, 122.24,121.97,121.59,120.42,114.47,109.89,47.70,44.92,10.84,4.18.HRMS(ESI)calcd for C 22 H 21 N 4 O,[M+H] + :m / z357.171, found 357.1712.

[0098] Example 9

[0099] The structure of compound 7D is shown below:

[0100]

[0101] Using 1-bromo-3-methyl-2-butene to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2, and other conditions remained unchanged, the experimental procedures of Example 1 were repeated to obtain compound 7D as a white solid with a yield of 38.2%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,t,J=5.8Hz),8.94(1H,s),8.74(1H,s),8.63–8.59(1H,m),8.20(1H,d ,J=7.8Hz),7.65(1H,td,J=7.7,1.7Hz),7.63–7.57(1H,m),7.46(1H,d,J=8.2Hz),7.39(1H ,d,J=7.8Hz),7.32(1H,t,J=7.5Hz),7.18(1H,dd,J=7.5,4.9Hz),5.28(1H,ddd,J=8.2,5.9 ,1.5Hz), 4.95(2H,d,J=6.7Hz), 4.88(2H,d,J=5.7Hz), 1.93(3H,s), 1.72(3H,d,J=1.6Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.78,157.64,149.29,141.48,139.90,137.55,136.76,136.63,130.38,129.05,128.67,122. 25,122.23,121.95,121.66,120.40,118.86,114.47,109.84,44.92,41.67,25.62,18.25.HRMS(ESI)calcd for C 23 H 22 N 4 ONa,[M+Na] + :m / z 393.1686, found 393.1675.

[0102] Example 10

[0103] The structure of compound 7F is shown below:

[0104]

[0105] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced by 2,3,6-trifluorobenzyl bromide. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 7F as a white solid with a yield of 67.9%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,t,J=5.8Hz),8.94(2H,d,J=12.1Hz),8.65–8.60(1H,m) ,8.21(1H,d,J=7.7Hz),7.71–7.61(3H,m),7.41(1H,d,J=7.8Hz),7.37(1H, ddd,J=8.0,6.2,1.8Hz),7.26(1H,s),7.21(1H,dd,J=7.2,4.6Hz),7.12(1H ,dd,J=9.1,5.1Hz),6.90–6.84(1H,m),5.65(2H,s),4.88(2H,d,J=5.7Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.59,157.50,149.20,141.38,140.58,137.63,136.92,130.53 ,130.51,130.48,129.51,129.04,122.30,122.23,122.07,121.84,121.01,1 17.66,117.59,117.53,117.46,114.38,113.93,113.82,113.69,111.49,111 .45,111.42,111.32,111.29,111.25,109.90,44.86,35.76.HRMS(ESI)calcd forC 25 H 18 F 3 N 4 O,[M+H] + :m / z 447.1427,found447.1430.

[0106] Embodiment 11

[0107] The structure of compound 7G is shown below:

[0108]

[0109] Using 4-fluoro-3-trifluoromethylbenzyl bromide to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2, and repeating the experimental procedures of Example 1 with other conditions unchanged, compound 7G was obtained as a white solid with a yield of 69.2%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,s),8.95(1H,d,J=0.9Hz),8.67(1H,d,J=0.9Hz),8.58 (1H,dt,J=4.9,1.3Hz),8.22(1H,d,J=7.9Hz),7.68–7.58(2H,m),7.50(1H ,dd,J=6.5,2.3Hz),7.41(1H,d,J=8.3Hz),7.37(2H,dt,J=7.1,3.4Hz),7. 21–7.14(2H,m),7.07(1H,t,J=9.3Hz),5.59(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.43,157.37,149.29,141.49,140.85,137.58,136.79,132.46,132.44,131.79,131.74,130.02,129.54,129.29,125.3 1,125.27,123.12,122.53,122.29,121.97,121.83,121.32,121.21,117.86,117.72,114.57,109.58,46.10,44.85.HRMS(ESI)calcd for C 26 H 19 F 4 N 4 O,[M+H] + :m / z 479.149, found 479.1490.

[0110] Example 12

[0111] The structure of compound 7H is shown below:

[0112]

[0113] Using 4-trifluoromethoxybenzyl bromide to replace 4-tert-butylbenzyl bromide used in step (6) of Example 2, and repeating the experimental procedures of Example 1 with other conditions unchanged, compound 7H was obtained as a white solid with a yield of 45.4%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,t,J=5.7Hz),8.96(1H,d,J=0.9Hz),8.68(1H,d,J=0.9Hz),8.58(1H,dt,J=4.8,1.4Hz),8.22(1H,d,J=7.8Hz),7.64(1H, td,J=7.7,1.8Hz),7.59(1H,ddd,J=8.3,7.1,1.2Hz),7.44(1H,s),7.40–7.33(2H,m),7.20–7.09(5H,m),5.57(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.53,157.45,149.27,148.85,141.63,140.61,137.68,136.79,134.74,130.25,129.39,129.14,127.8 6,122.43,122.27,121.97,121.76,121.54,121.23,121.01,119.52,114.54,109.75,46.37,44.86.HRMS(ESI)calcd forC 26 H 19 F 3 N 4 O 2 Na,[M+Na] + :m / z 499.1352, found 499.1370.

[0114] Example 13

[0115] The structure of compound 7I is shown below:

[0116]

[0117] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced by 3,5-dimethoxybenzyl bromide. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 7I as a white solid with a yield of 63.8%. 1 H NMR (CDCl 3,600MHz)δ8.98(2H,d,J=4.9Hz),8.74(1H,s),8.63–8.59(1H,m),8.25(1H,d,J=7. 8Hz),7.70–7.65(1H,m),7.63–7.57(1H,m),7.48(1H,d,J=8.3Hz),7.41(1H,d,J=7 .8Hz),7.36(1H,t,J=7.5Hz),7.26(1H,s),7.21(1H,dd,J=7.2,5.1Hz),6.34(1H,t ,J=2.3Hz),6.28(2H,d,J=2.2Hz),5.54(2H,s),4.88(2H,d,J=5.7Hz),3.68(6H,s). 13 C NMR (CDCl 3 ,151MHz)δ165.68,161.39,157.52,149.14,141.95,140.40,138.48,138.00,136.95,130.50,129.34,129.01, 122.38,122.30,122.09,121.76,120.83,114.55,109.94,104.67,99.25,55.31,47.29,44.82.HRMS(ESI)calcd for C 27 H 25 N 4 O 3 ,[M+H] + :m / z453.1921, found 453.1924.

[0118] Embodiment 14

[0119] The structure of compound 10e is shown below:

[0120]

[0121] The 4-cyanobenzyl bromide used in step (7) of Example 1 was replaced by bromomethylcyclopropane. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 10e as a white solid with a yield of 41.8%. 1 H NMR (CDCl 3,600MHz)δ9.00(1H,t,J=5.6Hz),8.90(1H,dd,J=4.1,1.0Hz),8.82(1H,dd,J=16.9,1.0Hz),8. 63(1H,ddd,J=5.0,1.8,0.9Hz),8.17(1H,dd,J=3.6,2.0Hz),7.69(1H,td,J=7.6,1.8Hz),7.59( 1H,ddd,J=10.7,8.7,2.0Hz),7.41(1H,d,J=9.3Hz),7.26(1H,s),7.22(1H,ddd,J=6.3,5.1,1.1 Hz), 4.88 (2H, d, J = 5.7Hz), 4.32 (1H, d, J = 6.6Hz), 3.98 (1H, s), 1.25 (1H, s), 0.68–0.40 (3H, m). 13 C NMR (CDCl 3 ,151MHz)δ165.49,165.46,157.47,149.23,140.46,140.43,140.30,14 0.05,138.53,138.23,136.91,130.75,130.38,129.06,128.99,128.09, 127.99,126.09,122.72,122.59,122.31,122.10,121.89,121.86,114.60,114.55,111.01,110.63,47.98,44.86,10.82,4.24.HRMS(ESI)calcd for C 22 H 19 C1N 4 ONa,[M+Na] + :m / z 413.1139, found 413.1147.

[0122] Embodiment 15

[0123] The structure of compound 10f is shown below:

[0124]

[0125] The 4-cyanobenzyl bromide used in step (7) of Example 1 was replaced by 2,3,6-trifluorobenzyl bromide. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 10f as a white solid with a yield of 68.9%. 1 H NMR (CDCl 3,600MHz)δ8.99(1H,t,J=5.7Hz),8.95(1H,s),8.87(1H,d,J=0.9Hz),8.63 (1H,dt,J=4.9,1.3Hz),8.14(1H,t,J=1.3Hz),7.68(1H,td,J=7.6,1.8Hz) ,7.58(2H,d,J=1.2Hz),7.41(1H,s),7.24–7.19(1H,m),7.13(1H,dd,J=9. 2,5.1Hz),6.88(1H,dd,J=3.6,2.2Hz),5.63(2H,s),4.88(2H,d,J=5.6Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.27,157.34,149.17,140.92,139.65,137.91,136.95,130.89,1 30.86,129.26,128.55,126.73,122.94,122.36,122.33,122.09,121.80,117.8 7,117.80,117.74,117.68,114.41,113.59,113.49,113.45,111.58,111.55,1 11.51,111.42,111.39,111.09,111.07,111.04,44.81,35.89.HRMS(ESI)calcd forC 25 H 17 CIF 3 N 4 O,[M+H] + :m / z481.1038, found 481.1040.

[0126] Example 16

[0127] The structure of compound 10g is shown below:

[0128]

[0129] 3,5-dimethoxybenzyl bromide was used to replace 4-cyanobenzyl bromide used in step (7) of Example 1. The other conditions remained unchanged and the experimental operation of Example 1 was repeated to obtain 10 g of compound as a white solid with a yield of 46.8%. 1 H NMR (CDCl 3,600MHz)δ8.97(1H,s),8.88(1H,d,J=0.9Hz),8.72(1H,d,J=1.0Hz),8.59(1H,ddd, J=4.9,1.8,0.9Hz),8.14(1H,d,J=2.0Hz),7.65(1H,td,J=7.6,1.8Hz),7.51(1H,dd ,J=8.7,2.1Hz),7.37(2H,d,J=8.9Hz),7.18(1H,ddd,J=7.6,4.9,1.1Hz),6.34(1H, t,J=2.3Hz), 6.22(2H,d,J=2.2Hz), 5.48(2H,s), 4.86(2H,d,J=5.7Hz), 3.66(6H,s). 13 CNMR (CDCl 3 ,151MHz)δ165.31,161.42,157.43,149.32,140.72,140.13,138.23,138.04,136.75,130.83,129.20,128.26, 126.42,122.75,122.25,121.93,121.88,114.51,111.11,104.60,99.28,55.30,47.37,44.88.HRMS(ESI)calcd for C 27 H 24 C1N 4 O 3 ,[M+H] + :m / z487.1532,found487.1530.

[0130] Embodiment 17

[0131] The structure of compound 10h is shown below:

[0132]

[0133] N-bromosuccinimide was used to replace N-chlorosuccinimide used in step (4) of Example 1, and 4-tert-butylbenzyl bromide was used to replace 4-cyanobenzyl bromide used in step (7) of Example 1. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 10h as a white solid with a yield of 76.9%. 1H NMR (Chloroform-d, 600MHz) δ9.19(1H,d,J=1.0Hz),8.99(1H,d,J=1.0Hz),8.77(1H,d,J=1.9Hz),7.82(1H,d,J=8.8Hz),7.80– 7.74(2H,m),7.37(1H,d,J=7.8Hz),7.33–7.28(3H,m),7.15(2H,d,J=8.4Hz),5.83(2H,s),4.69(2H,d,J=5.9Hz),1.20(10H,s). 13 C NMR (151MHz, DMSO-d6) δ165.16,158.70,150.58,149.29,140.88,140.59,138.15,137.23,134.37,132.42,131.88,130.12,127. 73,127.11,125.99,125.71,123.31,122.58,121.54,115.11,113.46,113.15,46.42,44.71,34.66,31.48.HRMS(ESI)calculated for C 27 H 23 Bn 4 O 3 ,[M+H] + :m / z 531.1027,found531.1014.

[0134] Embodiment 18

[0135] The structure of compound 10k is shown below:

[0136]

[0137] N-bromosuccinimide was used to replace the N-chlorosuccinimide used in step (4) of Example 1. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 10k as a white solid with a yield of 43.8%. 1 HNMR (CDCl 3,600MHz)δ8.97(1H,t,J=5.6Hz),8.87(1H,d,J=0.9Hz),8.66(1H,d,J=0.9Hz),8.59(1H,dt,J=4.7,1.3Hz),8.30(1H,d,J=1.8Hz),7.67 (2H,td,J=8.8,8.2,1.8Hz),7.59–7.55(2H,m),7.38(1H,s),7.29–7.25(1H,m),7.22–7.15(3H,m),5.64(2H,s),4.85(2H,d,J=5.6Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.06,157.20,149.28,141.26,140.93,140.09,137.73,136.84,132.95,132.09,130.41,128.39,1 27.02,125.17,123.45,122.35,122.01,118.18,114.59,114.12,112.27,111.17,46.83,44.81.HRMS(ESI)calcd for C 26 H 19 Bn 5 O,[M+H] + :m / z 496.0768, found 496.0776.

[0138] Embodiment 19

[0139] The structure of compound 101 is shown below:

[0140]

[0141] N-bromosuccinimide was used to replace N-chlorosuccinimide used in step (4) of Example 1, and bromomethylcyclopropane was used to replace 4-cyanobenzyl bromide used in step (7) of Example 1. The other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain compound 101 as a white solid with a yield of 31.8%. 1 H NMR (CDCl 3,600MHz)δ8.99(1H,s),8.86(1H,s),8.79(1H,s),8.62(1H,dd,J=4.8,1. 6Hz),8.28(1H,d,J=1.9Hz),7.67(2H,ddt,J=7.9,3.6,1.9Hz),7.39(2H,d ,J=17.8Hz),7.20(1H,dd,J=7.5,5.0Hz),4.87(2H,d,J=5.7Hz),4.28(2H, d,J=6.6Hz),1.33–1.28(1H,m),0.63–0.58(2H,m),0.42(2H,d,J=5.1Hz). 13 C NMR (CDCl 3 ,151MHz)δ165.42,157.48,149.29,140.30,137.97,136.81,131.53,130.70,127.87,124.85, 123.23,122.28,122.01,114.48,113.23,111.41,47.91,44.89,10.79,4.23.HRMS(ESI)calcd for C 22 H 20 Bn 4 O,[M+H] + :m / z435.0815,found435.0820.

[0142] Embodiment 20

[0143] The structure of compound 7e is shown below:

[0144]

[0145] 4-dimethylaminopyridine was used to replace 1-hydroxybenzotriazole used in step (5) of Example 2, and 2-chloro-5-hydroxymethylpyridine was used to replace 2-aminomethylpyridine used in step (5) of Example 2. Other conditions remained unchanged and the experimental procedures of Example 1 were repeated to obtain 62 mg of compound 7e as a white solid. The yield was 46%. 1 H NMR (CDCl 3,600MHz)δ8.91(2H,d,J=11.8Hz),8.57(1H,d,J=2.4Hz),8.23(1H,d,J=7.9Hz),7.87(1H,dd,J=8.2,2.5Hz),7.54(1H ,d,J=8.3Hz),7.40–7.33(2H,m),7.29(2H,d,J=8.3Hz),7.09(2H,d,J=8.2Hz),5.60(2H,s),5.49(2H,s),1.25(9H,s). 13 C NMR (CDCl 3 ,151MHz) δ165.79,151.52,151.27,150.07,141.92,139.42,138.11,136.96,132.65,132.29,130.82,129.26,128. 74,126.37,126.00,124.30,122.19,121.45,121.12,118.16,110.33,63.71,47.03,34.54,31.24.HRMS(ESI)calcd for C 29 H 27 C1N 3 O 2 ,[M+H] + :m / z 484.1787, found 484.1786.

[0146] The following are the pharmacological activity test results of the compounds prepared by the present invention:

[0147] Experimental instruments: clean bench (Shanghai Lichen Instrument Technology Co., Ltd.), constant temperature incubator (Thermoelectron Corporation), microplate reader (Thermo Fisher Scientific), inverted biological microscope (OLYMPUS).

[0148] Experimental reagents: cell culture medium RPMI-1640, DMEM (GIBCO), fetal bovine serum (Hangzhou Sijiqing Co., Ltd.), CCK-8 (Biosharp), DMSO (Sigma), RHKK-Ac-AMC (Shanghai Jier Biochemical Co., Ltd.), NAD + (Sigma), trypsin (Shanghai Aladdin Biochemical Technology Co., Ltd.), nicotinamide (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0149] Cell lines: human breast cancer cells MCF-7, human pancreatic cancer cells BxPC-3, Panc-1, MIA-PaCa-2, human leukemia cells HL-60, human neuroblastoma cells SH-SY5Y.

[0150] SIRT6 deacetylation inhibitory activity test method:

[0151] SIRT6 deacetylation activity was analyzed by Fluor-De-Lys (FDL) assay. 5 μM recombinant SIRT6 protein, 2.5 mM NAD + , 75 μM RHKK-Ac-AMC, DMSO or compound, and SIRT6 detection buffer was added to make up the total volume to 100 μL. The reaction mixture was incubated at 37 °C for 2 h, and then 100 μL of reaction stop solution (6 μg / μL trypsin and 40 mM nicotinamide) was added and further incubated at 37 °C for 2 h. The fluorescence intensity was measured using a microplate reader at 360 nm excitation and 460 nm emission. The IC of the derivatives was calculated using GraphPad Prism 8.0 50 value.

[0152] Cytostatic activity assay method:

[0153] The cells were maintained at 37°C and 5% CO 2 The cells were routinely cultured in a saturated humidity incubator. The cells in the logarithmic growth phase were digested with trypsin cell digestion solution (0.25% trypsin + 0.01% EDTA), centrifuged, resuspended, counted, and prepared into a concentration of 8×10 5 The cell suspension of 100 μL / mL was inoculated into a 96-well plate (100 μL per well) and placed in a constant temperature CO 2 Culture in an incubator overnight. After the cells adhere, use the culture medium to dilute the above-mentioned compound drugs to the required concentration, and add 100 μL of the corresponding drug-containing culture medium of different concentrations to each well. Set up three sub-wells for each group of drugs, and set up a blank control (containing only cell culture medium), a negative control (DMSO group) and a positive control group (paclitaxel group). Put the 96-well plate with the drug in the incubator and culture it for 24 hours. After adding 10 μL CCK-8 to each well of the 96-well plate, continue to culture in the incubator for 1 hour, then use an enzyme reader to test the OD value of each well at a wavelength of 450nm, and calculate the inhibition rate.

[0154] Inhibition rate calculation formula:

[0155]

[0156] Relative OD value of the experimental group = absolute OD value of the experimental group - absolute OD value of the blank control well

[0157] Experimental results:

[0158] Some of the compounds prepared by the present invention can effectively inhibit the deacetylation activity of recombinant SIRT6 (Table 1 and Table 2), among which compound 10d showed the strongest SIRT6 deacetylation inhibitory effect (IC 50 =5.81 μM).

[0159] Table 1 Inhibitory activity of the β-carboline disubstituted compounds prepared by the present invention on the deacetylation activity of SIRT6

[0160]

[0161]

[0162]

[0163] Table 2 Inhibitory activity of the β-carboline trisubstituted compounds prepared by the present invention on the deacetylation activity of SIRT6

[0164]

[0165]

[0166] The compound 10d prepared by the present invention has certain anticancer activity against human breast cancer cells MCF-7, human pancreatic cancer cells MIA-PaCa-2, human leukemia HL-60, and human neuroblastoma SH-SY5Y (Table 3)

[0167] Table 3 IC of compound 10d prepared in the present invention against cancer cells 50 Value (μM)

[0168]

[0169] The above pharmacological tests show that the target derivatives of the present invention have good SIRT6 inhibitory activity and certain anti-tumor cell proliferation activity, and have the potential to be used as tool compounds for SIRT6 function research and anticancer drugs.

Claims

1. A β-carboline 3,6,9-modified derivative or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula of the β-carboline 3,6,9-position modified derivative is shown below: in, X is O or NH, n is 1 or 2; R 1 is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the heterocyclic ring in the heteroaryl contains 1-4 N, O or S atoms, and the substituent in the substituted heteroaryl is a halogen, or a hydroxyl, or an alkyl group containing 1-7 carbon atoms; R 2 is an aryl or substituted aryl group containing 4-13 carbon atoms, or a hydrocarbon group containing 1-10 carbon atoms, or a cycloalkyl group containing 3-5 carbon atoms, or an alkoxy group containing 1-4 carbon atoms, and the substituent of the substituted aryl group is halogen, or cyano, or trifluoromethyl, or methoxy, or trifluoromethoxy, or an alkyl group containing 1-8 carbon atoms; R 3 is hydrogen or a halogen.

2. The β-carboline 3, 6, 9-modified derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X is NH, n is 1 or 2; R 1 is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the heterocyclic ring of the heteroaryl contains 1, 2 or 3 heteroatoms of N, O or S atoms, and the substituent in the substituted heteroaryl is a halogen, or a hydroxyl, or an alkyl group containing 1 to 4 carbon atoms; R 2 is phenyl or substituted phenyl, or naphthyl, or alkenyl containing 2-4 carbon atoms, or cycloalkyl containing 3 or 4 carbon atoms, or alkoxy containing 2 or 3 carbon atoms, wherein the substituent in the substituted phenyl is halogen, or cyano, or trifluoromethyl, or methoxy, or trifluoromethoxy, or alkyl containing 1-4 carbon atoms; R 3 It is a hydrogen, a chlorine, or a bromine atom.

3. The β-carboline 3,6,9-modified derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: X is NH, n is 1 or 2; R 1 is pyridyl, furyl, thienyl, triazolyl, pyrazinyl, pyrimidinyl, thiazolyl, wherein the substituent is halogen, or hydroxyl, or methyl; R 2 is phenyl or substituted phenyl, or naphthyl, or isobutylene, or cyclopropane, or ethoxy, wherein the substituent in the substituted aryl is a fluorine atom, or a chlorine atom, or a cyano group, or a trifluoromethyl group, or a methoxy group, or a trifluoromethoxy group, or a tert-butyl group; R 3 It is a hydrogen, a chlorine, or a bromine atom.

4. The 3,6,9-modified derivative of β-carboline or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: X is NH, n is 1; R 1 is pyridyl; R 2 is a substituted phenyl group, wherein the substituent is a cyano group; R 3 A chlorine atom.

5. The β-carboline 3, 6, 9-modified derivative and its pharmaceutically acceptable salt according to any one of claims 1 to 4, characterized in that: The structural formula of the β-carboline 3,6,9-modified derivative is shown below:

6. A method for preparing a β-carboline 3, 6, 9-position modified derivative represented by the general formula according to any one of claims 1 to 4, characterized in that: The steps include: (1) Compound 1 L-tryptophan was dissolved in a sodium hydroxide solution, and a 37% formaldehyde solution was added and reacted at 37°C for 2 days to obtain compound 2; compound 2 was dissolved in anhydrous methanol, thionyl chloride was added dropwise under an ice bath condition, and refluxed at 70°C for 6 hours to obtain compound 3; compound 3 was dissolved in N,N-dimethylformamide solution, potassium permanganate was added under an ice bath condition, and the reaction was transferred to room temperature for 14 hours to obtain compound 4; The compounds 1, 2, 3 and 4 have the following structures respectively: (2) Compound 4 was dissolved in methanol, 1N NaOH solution was added, and the mixture was heated at 78°C for 2 h to obtain compound 5; compound 5 was dissolved in N,N-dimethylformamide solution, carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding amino group were added. The compound was obtained by reacting at room temperature for 12 hours. Or add carbodiimide hydrochloride, 4-dimethylaminopyridine and the corresponding alcohol Compound Dissolve the above compound in tetrahydrofuran solution containing sodium hydride, stir for 1 hour, then add the corresponding bromohydrocarbon or benzyl bromide. React at room temperature for 12 hours to obtain the compound or The compound 5 has the following structure:

7. A method for preparing a β-carboline derivative modified at the 3, 6, and 9 positions as shown in any one of claims 1 to 4, characterized in that: The steps include: (1) Compound 1 L-tryptophan was dissolved in a sodium hydroxide solution, and a 37% formaldehyde solution was added and reacted at 37°C for 2 days to obtain compound 2; compound 2 was dissolved in anhydrous methanol, thionyl chloride was added dropwise under an ice bath condition, and refluxed at 70°C for 6 hours to obtain compound 3; compound 3 was dissolved in N,N-dimethylformamide solution, potassium permanganate was added under an ice bath condition, and the reaction was transferred to room temperature for 14 hours to obtain compound 4; The compounds 1, 2, 3 and 4 have the following structures respectively: (2) Compound 4 was dissolved in dichloromethane, a halogenating agent was added, and the mixture was refluxed at 78°C for 12 h to obtain compound The above compound was dissolved in methanol, 1N NaOH solution was added, and the mixture was heated at 78°C for 2h to obtain the compound The above compounds were dissolved in N,N-dimethylformamide solution, carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding amino The compound was obtained by reacting at room temperature for 12 hours. Or add carbodiimide hydrochloride, 4-dimethylaminopyridine and the corresponding alcohol Compound Dissolve the above compound in tetrahydrofuran solution containing sodium hydride, stir for 1 hour, then add the corresponding bromohydrocarbon or benzyl bromide. React at room temperature for 12 hours to obtain the compound or 8. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains a therapeutically effective amount of a β-carboline 3, 6, 9-position modified derivative represented by the general formula according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

9. Use of the 3, 6, 9-modified derivative of β-carboline according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 8 in the preparation of a SIRT6 inhibitor or in the preparation of a drug for treating a tumor disease.

10. The use according to claim 9, characterized in that: The tumor is breast cancer, pancreatic cancer, leukemia, neuroblastoma, liver cancer, and lung cancer.

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